Cyclodextrins (CDs) are generated through the action of cyclodextrin glucanotransferase (EC 2.4.1.19) (CGTase) on solubilized starch or related substrates containing 1 to 4-alpha glycosidic bonds. CDs are increasingly being utilized in several fields of science and technology to change inherent properties of a number of compounds such as vitamins, medicines, sanitary and agricultural effector-substances and aroma-compounds. Recently, similar applications have also been discovered for artificial, chemically modified cyclodextrins. The basis for the use of CDs lies in their ability to form molecular complexes or microcapsules with the effector compounds and thus to alter their physical and chemical properties such as solubility, volatility and absorption from the intestine.
The enzymatic reaction of CGTase predominantly yields a mixture of three forms of CDs, termed alpha, beta and gamma CDs. With many CGTases the beta form dominates in the conversion mixture. Because of its high concentration and low solubility in cold water, beta CD can be cheaply Produced in a pure crystalline state. Unfortunately, this is not the case with the less common forms of CDs. There is especially a need for pure, and considerably cheaper gamma CD than that presently available, because some important drugs would benefit from microencapsulation in CDs. These pharmaceuticals are, however, too large in molecular size to be imbibed into the internal cavity of other than gamma CD.
To produce gamma CD, processes have been devised, which use organic solvents such as trichloroethylene, tetrachloroethane or bromobenzene specifically to precipitate it, but these methods cannot be used when the product is intended for food or pharmaceutical use as it will frequently contain traces of the poisonous solvents.
Normally, 20-60% of the starch is converted to CDs depending on the concentration of starch being employed. When isolating beta CD, acyclic oligosaccharides must be converted to glucose or removed before the crystallization of beta CD is possible. However, usually when pure alpha or gamma CDs are desired the sugar fraction must be removed, its conversion to glucose is not adequate.
Separation of the alpha or gamma CDs out of CD mixtures is the most difficult step in their preparation. Two basically different approaches of the prior art using aqueous solvents are known. In the one method, CDs are separated by exclusion chromatography on gels such as Toyopearl.RTM., Molselect.RTM., or Sephadex.RTM. (Eur. pat. specification 45464; Die Starke 30 (1978) 276-279; Proceedings of the 1st Int. Symp. on Cyclodextrins, Budapest 1981, Ed. J. Szeijtli, pp. 61-68). This approach requires large columns, controlled temperature, and preliminary removal of small-molecular sugars and acyclic dextrins. The other approach is based on the ability of some commercially available, synthetic adsorption resins to adsorb CDs, provided the temperature of the column is strictly controlled. The earliest investigation was made by Yamamoto and Horikoshi (Die Starke 33, (1981) 244-246). Amberlite.RTM. XAD-4 adsorption resin was used, and the system was designed to purify alpha CD. The same principle for production also of gamma CD was later patented by Horikoshi et al. in 1984 (Eur.pat.specification 45464). Disadvantages of this method are that a stepwise temperature program is necessary, and that high temperatures (85.degree.-97.degree. C.) in the lengthy elution possibly give rise to chemical reactions. Additionally we have experimental evidence that CDs are adsorbed irreversibly onto the resins resulting in lowered adsorption capacity of the resin.
Adsorption chromatographic separation of glucose, maltose, and alpha and beta CDs on charcoal has also been reported (J. Lammers, J. Chromatogr. 41, 1969, pp. 462-466) but the support has low capacity for preparative purification of CDs.
The present invention includes an improved method of purifying individual CDs, especially gamma CD. Specific sorbents are synthesized for this purpose. The support matrix is primarily hydrophilic, which improves the swelling of the sorbents in water and increases the diffusional rates of CDs inside the porous network.